Current expectations for laboratory testing and adverse smallpox vaccine reactions

1
Current expectations for laboratory testing and
adverse smallpox vaccine reactions

• Department of Health and Human Services
• Centers for Disease Control and Prevention
• February 2003

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Poxviruses

• Two Subfamilies
• Chordopoxvirinae (vertebrate poxviruses)
• Orthopoxvirus (variola, vaccinia, cowpox,
  monkeypox, raccoonpox, camelpox, skunkpox,
  volepox, ectromelia, taterapox)
• Others
• Entomopoxvirinae (insect poxviruses)

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Characteristics of Orthopoxviruses

• Host ranges vary
• Variola vs vaccinia
• Antigenically similar serologic cross reactivity

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Febrile, vesicular rash illness algorithm for
evaluating patients for smallpox
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Differential Diagnosis

• Enteroviral infections (especially hand, foot and
  mouth)
• Disseminated herpes simplex
• Scabies, Insect bites
• Molluscum contagiosum (in immunocompromised)

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Vaccinia identification lab expectations/conside
rations

• Improve Public Health understanding of AEs and
  vaccination risks
• rarely a STAT function.
• LRN labs have means to detect vaccinia
• Real-time PCR test considered by the FDA an
  investigational device or a presumptive
  screening assay.
• Test results for pt management must be
  confirmed.
• Rule out other possible etiologies

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Specimen collection

• Specimen collection for skin lesion specimens
  associated with vaccination, of high suspicion
  for vaccinia
• http//www.bt.cdc.gov/agent/smallpox/vaccination/v
  accinia-specimen-collection.asp
• Specimen collection for suspect smallpox
  specimens (similar)
• www.bt.cdc.gov/agent/smallpox/r esponse-plan/files
  /guide-d.pdf

• http//

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Specimen collection kit
9

EM grids and grid box
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Specimen collection
Detailed description, with pictures on webcast of
December 5 and 6, 2002 To review http//www.bt.cd
c.gov/agent/smallpox/training/webcast/dec2002/inde
x.asp Go to Smallpox Vaccination Laboratory
Support module
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Lab methods for confirmation of orthopoxvirus
diagnosis

• PCR related methods for DNA identification, e.g.,
  real-time PCR, single gene PCR/RFLP, pangenomic
  methods if indicated
• Electron microscopy
• Histopathology
• Culture
• Serology?

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Laboratory Testing to Rule Out Other Rash Causing
Diseases

• VZV DFA, PCR, EM, Immunohistochemistry
• Herpes simplex PCR, EM, Immunohistochemistry and
  Culture
• Streptococcus, staphylococcus Gram stain, rapid
  tests, culture
• Enterovirus infections PCR, immunohistochemistry,
  culture

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Laboratory Testing to Rule Out Other Rash Causing
Diseases Continued

• Scabies Evidence of organisms
• Drug eruptions, allergic dermatitis skin biopsy,
  pathology
• Others as indicated from clinical impression
  (Sweets syndrome, Leukocytoclastic vasculitis,
  erythema multiforme)
• Biopsy for dermatopathologic examination

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Real-Time PCR assay (TaqMAN)E9L-Vaccinia
detection (Non-variola Eurasian orthopoxvirus
assay)

• Samples are tested using primers and probe
  designed to detect Eurasian Orthopoxvirus other
  than variola
• Potential human diseases detected
• Vaccinia
• Cowpox (Zoonotic disease of European origin)
• Monkeypox (Zoonotic disease of central Africa)

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Sensitivity of E9L vaccinia real-time PCR assay
during validation at LRN labs

• Limit of detection 5 to 50 genome copies during
  assay optimization
• 16/16 labs detected equivalent of 100 pfu
  vaccinia from dried, touch-prep slidevery
  sensitive!

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If smallpox were to re-emerge

• E9L test for vaccinia AEs would be modified to
  become test for variola virus DNA
• Alternate primer suppliedreal-time PCR test
  otherwise essentially the same
• Additional target(s) would also be used

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Sample requirements for Poxvirus DNA
identification

• Lesion roofs and crusts
• Vesicular fluids (touch prep)
• Biopsy, autopsy
• Others (e.g. CSF?)

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Negative Stain Electron Microscopy
vaccinia
1/2 hour per sample (for experienced
microscopist)
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Sample Requirements for Vaccinia (not variola)
Isolation

• Lesions roofs or crusts
• Vesicular fluids
• touch prep slide (reconstituted at lab)
• Frozen biopsy including PM tissue
• BSL-II conditions vaccinated lab workers
  preferable
• Variola referred to CDC (pre-event)

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Sample requirements for histopathology

• Biopsy or autopsy
• formalin fixed (not frozen)
• (remember to save fresh frozen bisected or
  duplicate sample for isolation)

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Where to Send Orthopox Specimens?

• Suspect vaccinia adverse events specimens that
  require identification of vaccinia go to closest
  Laboratory Response Network (LRN) laboratory.
• Contact your State Public Health Lab Director for
  shipping address
• All state and regional LRN labs can do real-time
  PCR for identification of vaccinia in AEs
• Specimens from persons with high suspicion of
  smallpox dx Refer to Rash, Vesicular Disease
  Algorithm. Contact State Public Health
  Laboratory director or Bioterrorism coordinator
  for shipping information and address.

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Specimen transport

• Standard dx specimen shipping guidelines
  available (subject to change)
  http//www.bt.cdc.gov/labissues/PackagingInfo.pdf
• Serum, if collected, should be refrigerated and
  shipped
• If spun and separated on site, freeze
• Formalin fixed material should be shipped at room
  temperature DO NOT FREEZE
• EM grids should be shipped at room temperature

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Specimen transport

• All other virus containing material should be
  stored and shipped frozen, unless it will be
  overnight shipped, then room temperature or
  refrigerated
• Keep all virus containing material out of direct
  sunlight

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Smallpox vs vaccinia Lab tests may be similar
but expectations for results and responses
different

• Pre-event smallpox dx implies massive public
  health response effort vaccination pt
  isolation and vaccination of contacts
• Vaccinia AEs expected to occur in small numbers
• AE patient care decisions based primarily on pt
  history and clinical considerations

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For More Information

• CDC Smallpox website
• www.cdc.gov/smallpox